1. Field of the Invention
This invention relates to a process for producing a high purity antihemophilic factor (AHF) concentrate having improved specific activity and reduced amount of fibrinogen impurity from an AHF-containing dispersion by subjecting the dispersion to a multi-step purification using conventional precipitants including aluminum hydroxide gel, polyethylene glycol, and glycine with sodium chloride.
2. Description of the Prior Art
Polson, U.S. Pat. No. 3,415,804, defines a 4-step process for fractionating a mixture of proteinaceous substances by
(1) mixing the proteinaceous substances with polyethylene glycol (PEG) in the presence of water to form a dispersion, the PEG-water dispersion being the only solvent phase present; PA0 (2) adjusting the relative concentrations of proteinaceous substances and remaining components in the mixture in step (1) to a pH and temperature to effect separation of a protein phase and an aqueous liquid phase containing water and the remaining components of the mixture; PA0 (3) separating said protein phase and said aqueous liquid phase from step (2); and PA0 (4) recovering a proteinaceous fractionation product from at least one of said fractions from step (3). PA0 (1) first with about 3-4% by weight of polyethylene glycol followed by recovery of the supernate; PA0 (2) then with polyethylene glycol added to about 10% by weight followed by recovery of the resulting precipitate; and PA0 (3) finally with 1.3-1.8 M glycine added to a solution of the precipitate from step (2) followed by recovery of the resulting precipitate. PA0 (a) isolating an antihemophilic factor containing starting material selected from plasma and a plasma fraction; PA0 (b) forming an aqueous solution of the isolated antihemophilic factor containing material from step (a), said solution optionally and preferably further having added thereto heparin or sodium heparin or mixtures thereof in the range of amounts of about 10-200 units/ml based on total volume of resulting solution, more preferably about 30-165 units/ml, especially preferably about 60 units/ml; PA0 (c) performing a first plasma protein precipitation by adding to the solution of the antihemophilic factor containing material from step (b) a suspension containing about 1-3% by weight, preferably about 2-3% by weight, based on total weight of suspension, of aluminum hydroxide in water and about 1-4% (w/v), based on volume of solution from step (b), of polyethylene glycol, and adjusting the pH of the resulting mixture to about 6.6-6.8 and the temperature of the resulting mixture to about 5.degree.-10.degree. C. and agitating the resulting mixture for about 15 minutes; PA0 (d) separating the precipitate and recovering the supernatant aqueous-polyethylene glycol solution resulting from step (c), for example, by a conventional centrifugation procedure; PA0 (e) performing a second plasma protein precipitation by adjusting the polyethylene glycol concentration of the aqueous-polyethylene glycol solution from step (d) to about 9-15% (w/v), based on volume of solution from step (d), for example, by adding sufficient polyethylene glycol or by subjecting the aqueous-polyethylene glycol solution from step (d) to ultrafiltration, and further adding to the so-adjusted solution about 10-20% by weight, based on total volume of solution in step (e), of glycine and about 10-20% by weight, based on total volume of solution in step (e), of sodium chloride, and adjusting the pH of the resulting mixture to about 5.7-6.8 and the temperature of the resulting mixture to about 5.degree.-10.degree. C. and agitating the resulting mixture for about 15 minutes; PA0 (f) separating the supernatant aqueous-polyethylene glycol solution and recovering the precipitate resulting from step (e), for example, by a conventional centrifugation procedure; PA0 (h) dissolving the washed precipitate from step (g) in a suitable aqueous medium, for example, a citrate-sodium chloride-glycine buffer solution; PA0 (i) subjecting the solution obtained in step (h) to sterile-filtration; and PA0 (j) freeze-drying the sterile-filtered solution from step (i) to obtain a dried, highly purified AHF product.
The polyethylene glycol suitable for use in the patented process has a molecular weight in the range of 300-100,000, preferably 600-20,000, more preferably 1,500-20,000, for example, 6,000. Polson also discloses well known variation of other parameters, such as, for example, to adjust the pH close to the isoelectric point of the protein component and to lower the ionic concentration and increase or lower temperature to increase separation of protein components, may be used in the patented process to have the same effect.
Shanbrom et al, U.S. Pat. No. 3,631,018, discloses an improved method of preparing a concentrate of AHF wherein the improvement is fractionating a cryoprecipitate concentrate of AHF with both polyethylene glycol and glycine in a three-step precipitation,
The polyethylene glycol suitable for use in the patented method has a molecular weight in the range of 200-20,000, preferably 400-6,000, more preferably about 4,000. The patent does not teach any ranges of limitations with respect to temperature and pH. However, Example 4 in the patent illustrates carrying out the precipitations at room temperature and at a pH of 6.5-6.88.
Johnson et al, U.S. Pat. No. 3,652,530, disclose a method of preparing highly purified AHF by treating an extract of a precipitate obtained by cryoethanol precipitation with polyethylene glycol in three successive precipitations, first with aluminum hydroxide gel at pH 5.6-7.0, then with polyethylene glycol to a concentration of 3.0-6.5%, and finally with added polyethylene glycol to a concentration of 10-12% to obtain a precipitate containing the highly purified AHF.
Fekete et al, U.S. Pat. No. 3,682,881, disclose a method for the preparation of a prothrombin complex and an AHF concentrate from citrated blood plasma treated with 1.5-1.8 M glycine. The resulting precipitate was treated successively with polyethylene glycol, first to a concentration of 3-4% and then 10% by weight, and finally with 1.8 M glycine.
Schwarz et al, U.S. Pat. No. 4,404,131, disclose an improved method of producing a F. VIII concentrate by subjecting a F. VIII concentrate obtained by conventional fractionation, e.g. cryoprecipitation, to cryoalcohol precipitation.
Fekete et al, U.S. Pat. No. Re. 29,698, disclose a method for improving the yield of AHF obtained from blood plasma and blood plasma fractions, obtained by cryoprecipitation, by the addition of heparin. The heparin-treated cryoprecipitate may then be further fractionated using polyethylene glycol and glycine. When the heparin-treated cryoprecipitate is further fractionated, heparin is preferably added twice, once to the initial cryoprecipitate and subsequently to the further fractionated concentrate.
Shanbrom, U.S. Pat. No. 4,069,216, discloses an improvement in the process disclosed in Shanbrom et al, U.S. Pat. No. 3,631,018 mentioned above, wherein the improvement is the step of holding a buffered solution of F. VIII and 6% polyol at 0.degree.-5.degree. C. until precipitation occurs.
Mitra et al, U.S. Pat. No. 4,386,068, disclose a process for producing an AHF concentrate by treating an aqueous suspension of cryoprecipitate containing AHF proteins with aluminum hydroxide gel, subjecting the resulting solution to ultrafiltration, and then constituting the solution resulting from the ultrafiltration in buffer and saline. Optionally, the solution resulting from the ultrafiltration may be treated with 1.6-2.2 M glycine for further purification.
Blomback et al, U.S. Pat. No. 4,348,315, disclose a process for purifying and/or concentrating the F. VIII complex, starting from cryoprecipitate or Cohn Fraction I-O, by dissolving a composition containing F. VIII together impurities in 1.5 M glycine solution at 15.degree. C. and pH 6.3-7.8 to obtain a solution containing F. VIII and a precipitate containing the impurities. Optionally, the patented process includes the additional step of adding PEG to the resulting F. VIII-containing glycine solution followed by precipitating and then concentrating purified F. VIII from the solution.
Rock, U.S. Pat. No. 4,203,891, discloses a method of increasing the yield of antihemophilic factor VIII (AHF), from whole blood, blood plasma or blood plasma fractions by collecting the blood or plasma or plasma fraction from a donor directly into an anticoagulant agent selected from heparin, sodium heparin, or mixtures thereof, which agent does not reduce the physiological concentration of calcium, and recovering the AHF. Preferably, the anticoagulant is used in the range of 0.1-10 units/ml based on total volume of whole blood or blood plasma and the AHF is recovered by fractionation using glycine, ethanol, ethanolglycine, polyethylene glycol or glycine-polyethylene glycol precipitation.
Rock et al, U.S. Pat. No. 4,289,691, discloses a method for obtaining AHF from fresh blood plasma by adding heparin, used in the range of about 1-10 units/ml of plasma, to fresh plasma collected by plasmapheresis into a calcium chelating anticoagulant, freezing the plasma, resolubilizing the plasma, isolating a cryoprecipitate from the plasma, resolubilizing the cryoprecipitate, adding a citrate saline heparin buffer to the resolubilized cryoprecipitate, incubating the resolubilized, buffered cryoprecipitate at about 0.degree.-10.degree. C. for a time in excess of about 1 hour in the presence of heparin precipitable cold insoluble globulin, separating an AHF rich precipitate and isolating AHF from the precipitate.
Amrani, U.S. Pat. No. 4,210,580, discloses a process for separating and isolating AHF and fibronectin from plasma by cryoprecipitation (0.degree.-15.degree. C.) in the presence of a sulfated mucopolysaccharide, e.g. heparin, to a concentration of about 0.15-0.25 mg/ml of plasma (approximately 22.5 to 37.5 units of heparin/ml of plasma). The resulting fibronectin precipitate is purified chromatographically and the heparin supernatant is mixed with an anion exchange resin such as DEAE cellulose with Heparasorb to remove heparin and to provide a supernatant having 90-95% of the original procoagulant activity.
Rock, U.S. Pat. No. 4,383,989, discloses a method of obtaining AHF by collecting freshly obtained plasma or plasma fractions directly into heparin, sodium heparin or mixtures thereof, in a proportion of about 6-8 units of heparin/ml of plasma, in the absence of a citrate buffer, and applying a cold incubation technique (0.degree.-10.degree. C.) using heparin precipitable cold insoluble globulin.
Fekete et al, U.S. Pat. No. Re. 29,698, discloses a method of improving the yield of AHF from plasma and plasma fractions by adding 0.01-10 units of heparin per ml of a concentrate of AHF obtained from the plasma or plasma fraction by cryoprecipitation.
Spain Pat. No. ES 8,307,101 (Derwent Abstract 84-025300/05) discloses a process to produce high purity AHF by the cold purification of cryoprecipitate, extraction of factor VIII in tris-hydrochloride buffer, deactivation of the prothrombin complex using heparin and selective precipitation of fibrinogen with polyethylene glycol. The resulting solution is treated with additional polyethylene glycol to obtain factor VIII containing cryoprecipitate at about 0.degree.-2.degree. C.
Although the processes for producing antihemophilic factor (also referred to as "AHF" and as "F. VIII") have provided some improvement in product quality, in terms of enhanced specific activity and purity, and also in yield or recovery of the product AHF, there remains a need for further process improvement to obtain an AHF concentrate in high yield and high purity with minimization of the reduction in AHF specific activity associated with prior art processes.